Structure-function analysis of inositol hexakisphosphate-induced autoprocessing of the Vibrio cholerae multifunctional autoprocessing RTX toxin

Katerina Prochazkova, Karla J. Fullner Satchell

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX toxin (MARTXVc). Autoprocessing of this toxin by an embedded cysteine protease domain (CPD) is essential for this toxin to induce actin depolymerization in a broad range of cell types. A homologous CPD is also present in the large clostridial toxin TcdB and recent studies showed that inositol hexakisphosphate (Ins(1,2,3,4,5,6)P6 or InsP6) stimulated the autoprocessing of TcdB dependent upon the CPD (Egerer, M., Giesemann, T., Jank, T., Satchell, K. J., and Aktories, K. (2007) J. Biol. Chem. 282, 25314-25321). In this work, the autoprocessing activity of the CPD within MARTXVc is similarly found to be inducible by InsP6. The CPD is shown to bind InsP6 (Kd, 0.6 μM), and InsP 6 is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 μM. Processed CPD did not bind InsP6 indicating that, subsequent to cleavage, the activated CPD may shift to an inactive conformation. To further pursue the mechanism of autoprocessing, conserved residues among 24 identified CPDs were mutagenized. In addition to cysteine and histidine residues that form the catalytic site, 2 lysine residues essential for InsP6 binding and 5 lysine and arginine residues resulting in loss of activity at low InsP6 concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP6 binding pocket and that the binding of InsP6 stimulates processing by altering the CPD to an activated conformation. After processing, InsP6 is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP6.

Original languageEnglish (US)
Pages (from-to)23656-23664
Number of pages9
JournalJournal of Biological Chemistry
Volume283
Issue number35
DOIs
StatePublished - Aug 29 2008

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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